Project Summary Rapid population ageing portends dramatic increases in the prevalence of adult-onset neurodegenerative disorders such as Parkinson?s disease (PD) in the coming decades. Inflammation, long considered an age- associated epiphenomenon of neurodegeneration, has gradually been recast as a causal factor through newly described neuroimmune disease mechanisms. Astrocytes are the predominant type of glial cell in the brain and are induced by inflammatory cytokines to adopt a neurotoxic A1 reactive phenotype, which my PhD thesis lab has linked to the degeneration of dopamine neurons in PD. However, the mechanisms by which A1 astrocytes contribute to early events in PD pathogenesis, such as the pathologic aggregation of ?-synuclein (?Syn), remain undefined. Understanding these processes will clarify the value of A1 astrocytes as a therapeutic target in early PD and may suggest novel roles in other neurodegenerative and neuroimmune disorders. My preliminary data show that A1 astrocytes exhibit robust induction of the NLRP3 inflammasome, an effector of the innate immune system recently implicated in ?Syn aggregation. Furthermore, I have shown that primary astrocyte cell cultures (1) can canonically activate the NLRP3 inflammasome, (2) readily internalize ?Syn, and (3) increase NLRP3 expression and IL-1? secretion in response to fibrillar ?Syn aggregates, consistent with inflammasome activation. Therefore, the central hypothesis of this proposal is that astrocyte NLRP3 inflammasome assembly can drive pathogenic ?Syn aggregation, which in turn reinforces NLRP3 activation and inflammation in PD. In Aim 1, we will determine whether the NLRP3 inflammasome promotes ?Syn aggregation in A1 astrocytes. In Aim 2, we will investigate astrocyte NLRP3 inflammasome activation by exogenous ?Syn fibrils. In Aim 3, we will demonstrate pathogenicity of astrocyte NLRP3 activation in the ?Syn preformed fibril (PFF) mouse model of PD. Primary astrocyte cultures will be used to establish the mechanistic plausibility of this model in vitro. We will use transgenic and gene-targeted mice to selectively delete NLRP3 in astrocytes and examine the impact on PD pathology in the ?Syn PFF mouse model. Our research will identify a novel mechanism by which A1 astrocytes contribute to early PD pathogenesis by promoting the aggregation of ?Syn into toxic species (Aim 1) and further show how ?Syn can activate the potent NLRP3 inflammatory cascade in astrocytes (Aim 2). We will also demonstrate that these processes have causal relevance in a well- characterized PD mouse model (Aim 3). Collectively, this project will enhance our understanding of astrocytes in PD etiology and characterize a novel disease role for the NLRP3 inflammasome. These studies comprise the scientific context of my training plan as an MD/PhD student at Johns Hopkins, which will facilitate my development as an independent physician-scientist. My sponsor, Ted Dawson, MD/PhD, is fully committed to supporting my success in this research and my long-term career-development.